Publications

• The high resolution crystal structure of human LCAT.

  Piper D.E., Romanow W.G., Gunawardane R.N., Fordstrom P., Masterman S., Pan O., Thibault S.T., Zhang R., Meininger D., Schwarz M., Wang Z., King C., Zhou M., Walker N.P.

  LCAT is intimately involved in HDL maturation and is a key component of the reverse cholesterol transport (RCT) pathway which removes excess cholesterol molecules from the peripheral tissues to the liver for excretion. Patients with loss-of-function LCAT mutations exhibit low levels of HDL cholest ... >> More

  LCAT is intimately involved in HDL maturation and is a key component of the reverse cholesterol transport (RCT) pathway which removes excess cholesterol molecules from the peripheral tissues to the liver for excretion. Patients with loss-of-function LCAT mutations exhibit low levels of HDL cholesterol and corneal opacity. Here we report the 2.65 Å crystal structure of the human LCAT protein. Crystallization required enzymatic removal of N-linked glycans and complex formation with a Fab fragment from a tool antibody. The crystal structure reveals that LCAT has an α/β hydrolase core with two additional subdomains that play important roles in LCAT function. Subdomain 1 contains the region of LCAT shown to be required for interfacial activation, while subdomain 2 contains the lid and amino acids that shape the substrate binding pocket. Mapping the naturally occurring mutations onto the structure provides insight into how they may affect LCAT enzymatic activity. << Less

  J. Lipid Res. 56:1711-1719(2015) [PubMed] [EuropePMC]

• Cloning and expression of human lecithin-cholesterol acyltransferase cDNA.

  McLean J., Fielding C., Drayna D., Dieplinger H., Baer B., Kohr W., Henzel W., Lawn R.

  cDNA and genomic cloning has been used to determine the mRNA and amino acid sequence of human plasma lecithin-cholesterol acyltransferase (LCATase; EC 2.3.1.43). The mature protein was found to contain 416 amino acid residues with a hydrophobic leader sequence of 24 amino acids. An unusual feature ... >> More

  cDNA and genomic cloning has been used to determine the mRNA and amino acid sequence of human plasma lecithin-cholesterol acyltransferase (LCATase; EC 2.3.1.43). The mature protein was found to contain 416 amino acid residues with a hydrophobic leader sequence of 24 amino acids. An unusual feature of the message is that the poly(A) signal AATAAA overlaps the COOH-terminal glutamic acid and stop codons, and the 3' untranslated region is only 23 bases. The protein itself is distinguished by a number of extended sequences of hydrophobic amino acids, one of which contains a hexapeptide identical with the interfacial binding segment of the active site of pancreatic lipase and is similar to the same site of lingual lipase. The cloned cDNA allows the expression of active LCATase by transfected tissue culture cells. << Less

  Proc. Natl. Acad. Sci. U.S.A. 83:2335-2339(1986) [PubMed] [EuropePMC]

• Apolipoprotein E is the major physiological activator of lecithin-cholesterol acyltransferase (LCAT) on apolipoprotein B lipoproteins.

  Zhao Y., Thorngate F.E., Weisgraber K.H., Williams D.L., Parks J.S.

  Our previous studies have indicated that lecithin-cholesterol acyltransferase (LCAT) contributes significantly to the apoB lipoprotein cholesteryl ester (CE) pool. Cholesterol esterification rate (CER) in apoA-I(-)(/)(-) apoE(-)(/)(-) mouse plasma was <7% that of C57Bl/6 (B6) mouse plasma, even th ... >> More

  Our previous studies have indicated that lecithin-cholesterol acyltransferase (LCAT) contributes significantly to the apoB lipoprotein cholesteryl ester (CE) pool. Cholesterol esterification rate (CER) in apoA-I(-)(/)(-) apoE(-)(/)(-) mouse plasma was <7% that of C57Bl/6 (B6) mouse plasma, even though apoA-I(-)(/)(-) apoE(-)(/)(-) plasma retained (1)/(3) the amount of B6 LCAT activity. This suggested that lack of LCAT enzyme did not explain the low CER in apoA-I(-)(/)(-) apoE(-)(/)(-) mice and indicated that apoE and apoA-I are the only major activators of LCAT in mouse plasma. Deleting apoE on low-density lipoprotein (LDL) reduced CER (1% free cholesterol (FC) esterified/h) compared to B6 (6% FC esterified/h) and apoA-I(-)(/)(-) (11% FC esterified/h) LDL. Similar sized LDL particles from all four genotypes were isolated by fast protein liquid chromatography (FPLC) after radiolabeling with [(3)H]-free cholesterol (FC). LDLs (1 microg FC) from each genotype were incubated with purified recombinant mouse LCAT; LDL particles from B6 and apoA-I(-)(/)(-) plasma were much better substrates for CE formation (5.7% and 6.3% CE formed/30 min, respectively) than those from apoE(-)(/)(-) and apoE(-)(/)(-) apoA-I(-)(/)(-) plasma (1.2% and 1.1% CE formed/30 min). Western blot analysis showed that the amount of apoA-I on apoE(-)(/)(-) LDLs was higher compared to B6 LDL. Adding apoE to incubations of apoA-I(-)(/)(-) apoE(-)(/)(-) very low density lipoprotein (VLDL) resulted in a 3-fold increase in LCAT CER, whereas addition of apoA-I resulted in a more modest 80% increase. We conclude that apoE is a more significant activator of LCAT than apoA-I on mouse apoB lipoproteins. << Less

  Biochemistry 44:1013-1025(2005) [PubMed] [EuropePMC]

• Chicken lecithin-cholesterol acyltransferase. Molecular characterization reveals unusual structure and expression pattern.

  Hengstschlaeger-Ottnad E., Kuchler K., Schneider W.J.

  Rapidly growing oocytes in the laying hen are, in addition to the liver, targets of the so-called "reverse cholesterol transport" (RCT) (Vieira, P.M., Vieira, A.V., Sanders, E.J., Steyrer, E., Nimpf, J., and Schneider, W.J. (1995) J. Lipid Res. 36, 601-610), pointing to the importance of this proc ... >> More

  Rapidly growing oocytes in the laying hen are, in addition to the liver, targets of the so-called "reverse cholesterol transport" (RCT) (Vieira, P.M., Vieira, A.V., Sanders, E.J., Steyrer, E., Nimpf, J., and Schneider, W.J. (1995) J. Lipid Res. 36, 601-610), pointing to the importance of this process in nonplacental reproduction. We have begun to delineate the details of this unique transport pathway branch by molecular characterization of the first nonmammalian lecithin-cholesterol acyltransferase (LCAT), the enzyme that catalyzes an early step in RCT. The biological significance of the enzyme is underscored by the high degree of protein sequence identity (73%) maintained from chicken to man. Interestingly, the conservation extends much less to the cysteine residues; in fact, two of the cysteines thought to be important in mammalian enzymes (residues 31 and 184 in man) are absent from the chicken enzyme, providing proof of their dispensability for enzymatic activity. Antibodies prepared against a chicken LCAT fusion protein cross-react with human LCAT and identify a 64-kDa protein present in enzymatically active fractions obtained by hydrophobic chromatography of chicken serum. The developmental and tissue distribution pattern of LCAT in females is striking; during embryogenesis and adolescence, LCAT expression is extremely high in liver but undetectable in brain. Upon onset of laying, however, brain LCAT mRNA increases suddenly and is maintained at levels 5 times higher than in liver, in stark contrast to most mammals. In adult roosters, the levels of LCAT transcripts in brain are lower than in liver. Together with the molecular characterization of chicken LCAT, these newly discovered developmental changes and gender differences in its expression establish the avian oocyte/liver system as a powerful model to delineate in vivo regulatory elements of RCT. << Less

  J. Biol. Chem. 270:26139-26145(1995) [PubMed] [EuropePMC]

• Comparative studies on the substrate specificity of lecithin:cholesterol acyltransferase towards the molecular species of phosphatidylcholine in the plasma of 14 vertebrates.

  Subbaiah P.V., Liu M.

  Comparative studies indicate that plasma cholesteryl ester (CE) composition is correlated with susceptibility to atherosclerosis. We previously showed that the lecithin:cholesterol acyltransferases (LCATs) of susceptible species such as rabbit, pig, and chicken (group I) differ in their substrate ... >> More

  Comparative studies indicate that plasma cholesteryl ester (CE) composition is correlated with susceptibility to atherosclerosis. We previously showed that the lecithin:cholesterol acyltransferases (LCATs) of susceptible species such as rabbit, pig, and chicken (group I) differ in their substrate and positional specificities from the LCATs of resistant species such as rat and mouse (group II). However, the relative importance of enzyme specificity and substrate phosphatidylcholine (PC) composition in determining the CE composition is not known. To address this, we analyzed the molecular species composition of plasma PC in the same 14 vertebrates in which we previously studied the CE composition and LCAT specificity. The utilization of native PC species by LCAT was studied by determining the loss of each PC after incubation of plasma at 37 degrees C. The major contributor for LCAT reaction was either 16:0-18:2 PC or 18:0-18:2 PC in all species except dog, in which it was 18:0-20:4 PC. The formation of 20:4 CE correlated more with the consumption of 18:0-20:4 PC in group I, and with the consumption of 16:0-20:4 PC in group II. The group II enzymes exhibited higher selectivity for sn-2-20:4 PCs, whereas the group I enzymes showed preference for sn-2-18:2 PCs. The synthesis of high percentage of 20:4 CE in dog plasma was found to be due to the presence of unusually high concentration of 18:0-20:4 PC, rather than due to enzyme selectivity. These results show that the PC molecular species composition, especially the concentrations of sn-2-20:4 phosphatidylcholines has profound influence on plasma CE composition, and possibly on atherogenic risk. << Less

  J. Lipid Res. 37:113-122(1996) [PubMed] [EuropePMC]

• Secretion of lecithin:cholesterol acyltransferase by brain neuroglial cell lines.

  Collet X., Francone O., Besnard F., Fielding C.J.

  The ability of different human and rat brain cell lines (neuronal and gliomal) to secrete lecithin:cholesterol acyltransferase (LCAT) was examined. Of these, the strongly secreting human gliomal (U343 and U251) cell lines were selected for a detailed study of enzymatic and structural properties of ... >> More

  The ability of different human and rat brain cell lines (neuronal and gliomal) to secrete lecithin:cholesterol acyltransferase (LCAT) was examined. Of these, the strongly secreting human gliomal (U343 and U251) cell lines were selected for a detailed study of enzymatic and structural properties of the secreted LCAT. Both plasma- and brain-derived enzymes are inhibited by DTNB (90%) and are activated by apolipoprotein A-I. LCAT mRNA was measured in these cell lines at levels similar to that found in HepG2 cells. In contrast, apoA-I, apoE, and apoD mRNAs were undetectable in these cell lines. The presence of functional LCAT secreted by cultured nerve cells provides an in vitro model to study the expression and function of LCAT in the absence of others factors of plasma cholesterol metabolism. << Less

  Biochem. Biophys. Res. Commun. 258:73-76(1999) [PubMed] [EuropePMC]

• Binding affinity and reactivity of lecithin cholesterol acyltransferase with native lipoproteins.

  Kosek A.B., Durbin D., Jonas A.

  The first step in the reaction of lecithin cholesterol acyltransferase (LCAT) with lipoproteins is the interfacial binding of the enzyme to the lipid surfaces. In this study the equilibrium dissociation constants (Kds) for the interaction of pure human plasma LCAT with LDL, HDL2, HDL3, and a recon ... >> More

  The first step in the reaction of lecithin cholesterol acyltransferase (LCAT) with lipoproteins is the interfacial binding of the enzyme to the lipid surfaces. In this study the equilibrium dissociation constants (Kds) for the interaction of pure human plasma LCAT with LDL, HDL2, HDL3, and a reconstituted discoidal HDL (rHDL) were determined by the activity-inhibition method. In addition, enzyme kinetics were measured with each of the lipoprotein substrates. Based on phospholipid concentrations, the Kd values (0.9 x 10(-5) to 4.6 x 10(-5) M) increased in the order rHDL = HDL3 </= HDL2 < LDL while the relative reactivities (app Vmax/app Km) with LCAT were 100, 16, 1, 6%, respectively, for the different lipoproteins. These quantitative measures were used to predict the distribution of LCAT in plasma and to explain cholesterol esterification when HDL are absent or ineffective as substrates. << Less

  Biochem. Biophys. Res. Commun. 258:548-551(1999) [PubMed] [EuropePMC]

• Negative charge at amino acid 149 is the molecular determinant for substrate specificity of lecithin: cholesterol acyltransferase for phosphatidylcholine containing 20-carbon sn-2 fatty acyl chains.

  Zhao Y., Wang J., Gebre A.K., Chisholm J.W., Parks J.S.

  We previously described a point mutation in human LCAT (E to A at residue 149; hE149A) that demonstrated greater activity with phosphatidylcholine (PC) substrate containing 20:4 in the sn-2 position compared with the wild-type enzyme [hLCAT; Wang et al. (1997) J. Biol. Chem. 272, 280-286], resulti ... >> More

  We previously described a point mutation in human LCAT (E to A at residue 149; hE149A) that demonstrated greater activity with phosphatidylcholine (PC) substrate containing 20:4 in the sn-2 position compared with the wild-type enzyme [hLCAT; Wang et al. (1997) J. Biol. Chem. 272, 280-286], resulting in a human enzyme with the substrate specificity similar to that of rat LCAT. The purpose of the present study was to explore the molecular basis for the role of amino acid 149 in determining fatty acyl substrate specificity. In the first experiment, the reverse mutation in rat LCAT (rA149E) converted substrate specificity of rat LCAT toward that of the human enzyme, demonstrating that the mutation was context independent and reversible. In the second experiment, we found that hE149A compared with hLCAT demonstrated higher activity with PC species containing 20-carbon, but not 18-carbon, sn-2 fatty acyl chains. The increased activity of hE149A was due to an increase in apparent V(max) but not to apparent K(m) or LCAT binding to the PC surface. Substitution of different amino acids in the 149 position of hLCAT showed that activation of the enzyme with sn-2 20:4 containing PC substrate was only observed when the negative charge at residue 149 was removed. We conclude that the negative charge at amino acid 149 of LCAT is a critical determinant for the specificity of the enzyme for PC containing 18- vs 20-carbon sn-2 fatty acyl chains. << Less

  Biochemistry 42:13941-13949(2003) [PubMed] [EuropePMC]

  This publication is cited by 12 other entries.

• LCAT synthesized by primary astrocytes esterifies cholesterol on glia-derived lipoproteins.

  Hirsch-Reinshagen V., Donkin J., Stukas S., Chan J., Wilkinson A., Fan J., Parks J.S., Kuivenhoven J.A., Lutjohann D., Pritchard H., Wellington C.L.

  Lipid trafficking in the brain is essential for the maintenance and repair of neuronal membranes, especially after neurotoxic insults. However, brain lipid metabolism is not completely understood. In plasma, LCAT catalyses the esterification of free cholesterol on circulating lipoproteins, a key s ... >> More

  Lipid trafficking in the brain is essential for the maintenance and repair of neuronal membranes, especially after neurotoxic insults. However, brain lipid metabolism is not completely understood. In plasma, LCAT catalyses the esterification of free cholesterol on circulating lipoproteins, a key step in the maturation of HDL. Brain lipoproteins are apolipoprotein E (apoE)-containing, HDL-like particles secreted initially as lipid-poor discs by glial cells. LCAT is synthesized within the brain, suggesting that it may play a key role in the maturation of these lipoproteins. Here we demonstrate that astrocytes are the primary producers of brain LCAT. This LCAT esterifies free cholesterol on nascent apoE-containing lipopoproteins secreted from glia. ApoE is the major LCAT activator in glia-conditioned media (GCM), and both the cholesterol transporter ABCA1 and apoE are required to generate glial LCAT substrate particles. LCAT deficiency leads to the appearance of abnormal approximately 8 nm particles in GCM, and exogenous LCAT restores the lipoprotein particle distribution to the wild-type (WT) pattern. In vivo, complete LCAT deficiency results in a dramatic increase in apoE-HDL and reduced apolipoprotein A-I (apoA-I)-HDL in murine cerebrospinal fluid (CSF). These data show that brain LCAT esterifies cholesterol on glial-derived apoE-lipoproteins, and influences CSF apoE and apoA-I levels. << Less

  J. Lipid Res. 50:885-893(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structure and function of lysosomal phospholipase A2 and lecithin:cholesterol acyltransferase.

  Glukhova A., Hinkovska-Galcheva V., Kelly R., Abe A., Shayman J.A., Tesmer J.J.

  Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of key lipid-metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underli ... >> More

  Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of key lipid-metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause fish eye disease and familial LCAT deficiency. Here we describe several high-resolution crystal structures of human LPLA2 and a low-resolution structure of LCAT that confirms its close structural relationship to LPLA2. Insertions in the α/β hydrolase core of LPLA2 form domains that are responsible for membrane interaction and binding the acyl chains and head groups of phospholipid substrates. The LCAT structure suggests the molecular basis underlying human disease for most of the known LCAT missense mutations, and paves the way for rational development of new therapeutics to treat LCAT deficiency, atherosclerosis and acute coronary syndrome. << Less

  Nat. Commun. 6:6250-6250(2015) [PubMed] [EuropePMC]